METHOD FOR CONSTRUCTING CORRELATION MODEL BETWEEN RELATIVE BIOAVAILABILITY AND BIOACCESSIBILITY OF CADMIUM IN EARTHWORM

Abstract

The present disclosure provides a method for constructing a correlation model between relative bioavailability and bioaccessibility of cadmium in earthworm, belonging to the technical field of cadmium detection in earthworms. The method includes the following steps: determination of relative bioavailability of cadmium in earthworms, determination of bioaccessibility of cadmium in the earthworm based on an in vitro physiologically-based extraction test (PBET) method, and construction of a correlation model between the relative bioavailability and the bioaccessibility of cadmium in the earthworm. The method enables to directly put the bioaccessibility obtained by the in vitro method into a regression equation to obtain the bioavailability of cadmium in subsequent studies. The method eliminates a need for animal experiments in follow-up researches, saves resources to the greatest extent, and provides a new idea for objectively and scientifically evaluating the health risk of cadmium in the earthworm to human bodies.

Claims

1. A method for constructing a correlation model between relative bioavailability and bioaccessibility of cadmium in earthworm, comprising the following steps: grouping mice, conducting successive administration for 6 days to 8 days, and sacrificing the mice; determining a cadmium content in liver, kidney, brain, and femur tissues of the mice, and calculating relative bioavailability of cadmium in earthworm; simulating gastrointestinal digestion by a physiologically-based extraction test (PBET) method and extracting digestive juices, and determining a residual amount of cadmium in gastric juice and intestinal juice separately to obtain bioaccessibility of cadmium in the earthworm; and constructing a correlation model according to the relative bioavailability and the bioaccessibility of cadmium in the earthworm to obtain a regression equation.

2. The method according to claim 1, wherein the grouping is conducted specifically as follows: the mice fed with normal saline are used as a blank control group, the mice fed with cadmium are used as a positive control group, and the mice fed with the earthworm are used as an earthworm group; and the positive control group is divided into a low-concentration positive control group, a medium-concentration positive control group, and a high-concentration positive control group.

3. The method according to claim 2, wherein the low-concentration positive control group has a cadmium concentration of 3.5 mg/kg to 4.5 mg/kg; the medium-concentration positive control group has a cadmium concentration of 18 mg/kg to 22 mg/kg; the high-concentration positive control group has a cadmium concentration of 95 mg/kg to 105 mg/kg; and the earthworm group has an earthworm concentration of 1.128 mg/kg to 7.401 mg/kg.

4. The method according to claim 1, wherein a calculation formula of the relative bioavailability is as follows: $\mathrm{Relative}\mathrm{bioavailability}=\frac{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{Earthworm}}}{\mathrm{Earthworm}\mathrm{concentration}}\times \frac{\mathrm{Positive}\mathrm{control}\mathrm{group}\mathrm{concentration}}{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{positive}}};$ wherein (liver+kidney+brain+femur).sub.Earthworm is the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 6 days to 8 days after administration in the earthworm group, (liver+kidney+brain+femur).sub.positive is the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 6 days to 8 days after administration in the low-concentration positive control group, the medium-concentration positive control group, and the high-concentration positive control group.

5. The method according to claim 1, wherein cadmium content in the tissues is determined by an inductively coupled plasma-mass spectrometry (ICP-MS) method; and the residual amount of cadmium in the gastric juice and the intestinal juice is determined by the ICP-MS method.

6. The method according to claim 1, wherein the administration is conducted by gavage.

7. The method according to claim 5, wherein working parameters of the ICP-MS method are as follows. TABLE-US-00006 Working Item Working Item parameter Plasma gas flow 15.0 L/min Peristaltic pump 0.20 r/s Sampling depth 10 mm Number of 1 Nebulization 2° C. Auxiliary 0.8 L/min chamber gas flow He gas flow 5 mL/min Carrier 0.8 L/min gas flow Radio- 1550 W Data Peak frequency sampling hopping power mode sampling Determination 6 Number 100 point/peak of scans Number of 3 repetitions

8. The method according to claim 1, wherein the regression equation is as follows:
relative bioavailability=1.09×bioaccessibility+6.97.

9. The method according to claim 6, wherein working parameters of the ICP-MS method comprise: a plasma gas flow is about 15.0 L/min; a sampling depth is about 10 mm; a nebulization chamber is at about 2° C.; an He gas flow is about 5 mL/min; a radio-frequency power is about 1550 W; a determination point/peak is about 6; a number of repetitions is about 3; a peristaltic pump is about 0.20 r/s; a second number of repetitions is about 1; an auxiliary gas flow is about 0.8 L/min; a carrier gas flow is about 0.8 L/min; a data sampling mode comprises peak hopping sampling; and a number of scans is about 100 times.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows results of relative bioavailability of cadmium in 10 batches of earthworm; and

[0022] FIG. 2 shows a correlation between bioavailability and bioaccessibility of cadmium in earthworm.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The present disclosure provides a method for constructing a correlation model between relative bioavailability and bioaccessibility of cadmium in earthworm, including the following steps:

[0024] grouping mice, conducting successive administration for 6 days to 8 days, and sacrificing the mice; determining a cadmium content in liver, kidney, brain, and femur tissues of the mice, and calculating relative bioavailability of cadmium in earthworm;

[0025] simulating gastrointestinal digestion by a physiologically-based extraction test (PBET) method and extracting digestive juice, and determining a residual amount of cadmium in gastric juice and intestinal juice separately to obtain bioaccessibility of cadmium in the earthworm; and

[0026] constructing a correlation model according to the relative bioavailability and the bioaccessibility of cadmium in the earthworm to obtain a regression equation.

[0027] In the present disclosure, the mice are grouped, administered continuously for 6 days to 8 days, and then sacrificed. Preferably, the grouping is conducted specifically as follows: the mice fed with normal saline are used as a blank control group, the mice fed with cadmium are used as a positive control group, and the mice fed with the earthworm are used as an earthworm group; and the positive control group is divided into a low-concentration positive control group, a medium-concentration positive control group, and a high-concentration positive control group. The low-concentration positive control group has a cadmium concentration of preferably 3.5 mg/kg to 4.5 mg/kg; the medium-concentration positive control group has a cadmium concentration of preferably 18 mg/kg to 22 mg/kg; the high-concentration positive control group has a cadmium concentration of preferably 95 mg/kg to 105 mg/kg; and the earthworm group has an earthworm concentration of preferably 1.128 mg/kg to 7.401 mg/kg, the administration is conducted preferably by gavage. More preferably, the grouping is conducted specifically as follows:

[0028] 1) control group: 6 mice are subjected to administration of a normal saline at 1 mL/mice by gavage, once a day;

[0029] 2) low-concentration positive control group: 6 mice are subjected to administration at 1 mL/mice by gavage with a Cd concentration of 4 mg/kg, once a day;

[0030] 3) medium-concentration positive control group: 6 mice are subjected to administration at 1 mL/mice by gavage with a Cd concentration of 20 mg/kg, once a day;

[0031] 4) high-concentration positive control group: 6 mice are subjected to administration at 1 mL/mice by gavage with a Cd concentration of 100 mg/kg, once a day; and

[0032] 5) earthworm group: 6 mice are subjected to administration at 1 mL/mice by gavage with an earthworm concentration of 5 mg/kg, once a day.

[0033] In the present disclosure, cadmium content in liver, kidney, brain, and femur tissues of the mice is determined, and relative bioavailability of cadmium in earthworm is calculated. Each tissue of the liver, kidney, brain, and femur is weighed, freeze-dried, and digested before determining cadmium content in the tissues. There is no special limitation on the method of freeze-drying and digestion, and methods known in the art can be used. The cadmium content in the tissues is determined preferably by an ICP-MS method. After the determination of cadmium content, relative bioavailability is calculated, where the calculation formula of the relative bioavailability is preferably as follows:

[00002]$\mathrm{Relative}\mathrm{bioavailability}=\frac{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{Earthworm}}}{\mathrm{Earthworm}\mathrm{concentration}}\times \frac{\mathrm{Positive}\mathrm{control}\mathrm{group}\mathrm{concentration}}{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{positive}}};$

where,

[0034] (liver+kidney+brain+femur).sub.Earthworm is the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 6 days to 8 days after administration of the earthworm group (unit: μg/g dry weight), (liver+kidney+brain+femur).sub.positive is a sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 6 days to 8 days after administration of the low-concentration positive control group, the medium-concentration positive control group, and the high-concentration positive control group (unit: μg/g dry weight); and the positive control group concentration or the earthworm concentration is calculated in μg/g body weight A combined exposure method in which liver, kidney, brain, and femur are used as an action target is used, such that the relative bioavailability determined is more stable.

[0035] In the present disclosure, gastrointestinal digestion is simulated by a PBET method and digestive juices are extracted, and a residual amount of cadmium in gastric juice and intestinal juice is determined to obtain bioaccessibility of cadmium in the earthworm. There is no special limitation on specific steps for simulating the gastrointestinal digestion by the PBET method and extracting the digestive juices, and methods known in the art can be used. The residual amount of cadmium in the gastric juice and the intestinal juice is preferably determined by the ICP-MS method.

[0036] The working parameters of the ICP-MS method are preferably as follows:

TABLE-US-00002 Working Item Working Item parameter Plasma gas flow 15.0 L/min Peristaltic pump 0.20 r/s Sampling depth 10 mm Number of 1 repetitions Nebulization 2° C. Auxiliary 0.8 L/min chamber gas flow He gas flow 5 mL/min Carrier gas flow 0.8 L/min Radio-frequency 1550 W Data Peak power sampling hopping mode sampling Determination 6 Number 100 point/peak of scans Number of 3 repetitions

[0037] The cadmium content in earthworm was determined using the ICP-MS working parameters, and a standard curve was drawn. According to an analysis of a fitting degree of the curve, a correlation coefficient of cadmium was determined to be 0.997, meaning a desirable linear relationship; and suitability of the method was confirmed using accompanying quality control, and results were in line with residue testing requirements.

[0038] In the present disclosure, a correlation model is constructed according to the relative bioavailability and the bioaccessibility of cadmium in the earthworm to obtain a regression equation. The regression equation is as follow: relative bioavailability=1.09×bioaccessibility+6.97, where r.sup.2=0.84.

[0039] In the present disclosure, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0040] The technical solutions are clearly and completely described below in conjunction with examples of the present disclosure. It is clear that the described examples are merely a part, rather than all of the examples of the present disclosure. All other examples obtained by a person of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.

EXAMPLE 1

[0041] A method for constructing a correlation model between relative bioavailability and bioaccessibility of cadmium in earthworm included the following steps:

[0042] (1) Determination of Relative Bioavailability of Cadmium in Earthworm

[0043] After adaptive feeding, 18 g to 22 g of female mice were randomly grouped (6 mice/group), and weighed after fasting overnight; 10 batches of earthworm groups were given multiple times of intragastric administration of earthworm; high-concentration, medium-concentration and low-concentration positive control groups were administered with corresponding concentrations of cadmium by gavage; meanwhile, mice administered with normal saline by gavage were used as a blank control group. The grouping and administration were conducted as follows:

[0044] 1) control group: 6 mice were subjected to administration of normal saline at 1 mL/mice by gavage;, once a day;

[0045] 2) low-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 4 mg/kg, once a day;

[0046] 3) medium-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 20 mg/kg, once a day;

[0047] 4) high-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 100 mg/kg, once a day; and

[0048] 5) earthworm group: 6 mice were subjected to administration at 1 mL/mice by gavage with a earthworm concentration of 5 mg/kg, once a day.

[0049] After 7 days of continuous administration, the mice were sacrificed, and liver, kidney, brain, and femur tissues of the mice were taken out, weighed, freeze-dried, and digested; a cadmium content of each tissue was determined by ICP-MS, and relative bioavailability was calculated; where the relative bioavailability was calculated according to the following formula:

[00003]$\mathrm{Relative}\mathrm{bioavailability}=\frac{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{Earthworm}}}{\mathrm{Earthworm}\mathrm{concentration}}\times \frac{\mathrm{Positive}\mathrm{control}\mathrm{group}\mathrm{concentration}}{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{positive}}}$

[0050] where, (liver+kidney+brain+femur).sub.Earthworm was the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 7 days after administration in the earthworm group (unit: μg/g dry weight), (liver+kidney+brain+femur)positive was the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 7 days after administration in the low-concentration positive control group, the medium-concentration positive control group, and the high-concentration positive control group (unit: μg/g dry weight).

[0051] A method for determining cadmium by ICP-MS included:

[0052] A single element standard solution of cadmium was precisely measured, and a series of solutions containing Cd at a concentration of 0.2, 0.5, 2, 4, and 10 μg/mL were prepared using a 5% HNO.sub.3 solution, and a standard curve was drawn. The working conditions of ICP-MS were shown in Table 1.

TABLE-US-00003 TABLE 1 Working parameters of ICP-MS instrument Working Item Working Item parameter Plasma gas flow 15.0 L/min Peristaltic pump 0.20 r/s Sampling depth 10 mm Number of 1 repetitions Nebulization 2° C. Auxiliary 0.8 L/min chamber gas flow He gas flow 5 mL/min Carrier 0.8 L/min gas flow Radio-frequency 1550 W Data Peak power sampling hopping mode sampling Determination 6 Number 100 point/peak of scans Number of 3 repetitions

[0053] According to an analysis of fitting degree of the curve, a correlation coefficient of cadmium was determined to be 0.997, with a desirable linear relationship.

[0054] (2) Bioaccessibility of Cadmium in Earthworm Based on In Vitro PBET Method

[0055] 1) Preparation of extracted gastric juice and investigation of a dissolution rate of cadmium in earthworm: Earthworm samples were crushed, sieved through a 50-mesh sieve to obtain earthworm powder. To 0.5 g of the earthworm powder was added 50 mL of simulated gastric juice to obtain a mixture. Argon was introduced for 1 min to 2 min to simulate an anaerobic environment in the gastrointestinal tract, the mixture was shaken at 100 r/min for 1 h in a constant-temperature water bath at 37° C., and centrifuged at 4,000 r/min for 5 min; 25 mL of a resulting supernatant was concentrated to 3 mL at low temperature on an electric hot plate, and 5 mL of nitric acid was added to the supernatant after cooling, followed by digestion in a microwave digestion apparatus; after cooling, a resulting digestion solution was diluted to a 50 mL volumetric flask with ultrapure water for testing; meanwhile, a reagent blank solution was prepared in the same way. The simulated gastric juice was prepared by mixing 1.25 g of pepsin, 0.5 g of sodium citrate, 0.5 g of sodium malate, 420 μL of lactic acid, and 500 μL of acetic acid, making up the volume to 1 L after dissolving, and adjusting pH to a value of 2.0 with hydrochloric acid.

[0056] 2) Preparation of extracted intestinal juice and investigation of a dissolution rate of cadmium in earthworm: after gastric juice extraction, 50 mL of a simulated intestinal juice was added to residues to obtain a mixture, the mixture was shaken at 100 r/min for 4 h in a constant-temperature water bath at 37° C., and centrifuged at 4,000 r/min for 5 min; 25 mL of a resulting supernatant was concentrated to 3 mL at low temperature on an electric hot plate, and 5 mL of nitric acid was added to the supernatant after cooling, followed by digestion in a microwave digestion apparatus; after cooling, a resulting digestion solution was diluted and made up to 50 mL in a volumetric flask with ultrapure water for testing; meanwhile, a reagent blank solution was prepared in the same way. The simulated intestinal juice was prepared by mixing 0.5 g of a pancreatic enzyme and 1.75 g of ammonium cholate, making up the volume to 1 L after dissolving, and adjusting pH to a value of 7.0 with NaOH.

[0057] After the extraction, the residual cadmium was determined in the extracted gastric juice and intestinal juice using the ICP-MS method in step (1) separately to obtain the bioaccessibility of cadmium in earthworm.

[0058] (3) A correlation model was constructed according to results of the relative bioavailability and the bioaccessibility to obtain a regression equation.

[0059] FIG. 1 shows relative bioavailability results of cadmium in 10 batches of earthworm determined in step (1).

[0060] The results in FIG. 1 show that the relative bioavailability of cadmium in 10 batches of earthworm is 13.5% to 80.5%.

[0061] FIG. 2 shows the correlation model of the bioavailability results and in vitro bioaccessibility results of cadmium in earthworm in step (3).

[0062] FIG. 2 shows that the regression equation is as follow: relative bioavailability=1.09×bioaccessibility+6.97, where r.sup.2=0.84.

EXAMPLE 2

[0063] A method for constructing a correlation model between relative bioavailability and bioaccessibility of cadmium in earthworm included the following steps:

[0064] (1) Determination of Relative Bioavailability of Cadmium in Earthworm

[0065] After adaptive feeding, female mice having a body weight of 18 g to 22 g were randomly grouped (6 mice/group), and weighed after fasting overnight; 10 batches of earthworm groups were given multiple doses of intragastric administration of earthworm; high-concentration, medium-concentration and low-concentration positive control groups were administered with corresponding concentrations of cadmium by gavage, separately; meanwhile, mice administered with normal saline by gavage were used as a blank control group. The grouping and administration were conducted as follows:

[0066] 1) control group: 6 mice were subjected to administration of normal saline at 1 mL/mice by gavage;, once a day;

[0067] 2) low-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 3.5mg/kg, once a day;

[0068] 3) medium-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 18mg/kg, once a day;

[0069] 4) high-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 95 mg/kg, once a day; and

[0070] 5) earthworm group: 6 mice were subjected to administration at 1 mL/mice by gavage with a earthworm concentration of 1.128 mg/kg, once a day.

[0071] After 8 days of continuous administration, the mice were sacrificed, and liver, kidney, brain, and femur tissues of the mice were taken out, weighed, freeze-dried, and digested; a cadmium content of each tissue was determined by ICP-MS, and relative bioavailability was calculated; where the relative bioavailability was calculated according to the following formula:

[00004]$\mathrm{Relative}\mathrm{bioavailability}=\frac{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{Earthworm}}}{\mathrm{Earthworm}\mathrm{concentration}}\times \frac{\mathrm{Positive}\mathrm{control}\mathrm{group}\mathrm{concentration}}{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{positive}}}$

[0072] where, (liver+kidney+brain+femur).sub.Pheretima was the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 8 d after administration in the earthworm group (unit: μg/g dry weight), (liver+kidney+brain 30 femur).sub.positive was the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 8 days after administration in the low-concentration positive control group, the medium-concentration positive control group, and the high-concentration positive control group (unit: μg/g dry weight).

[0073] The method for determining cadmium by ICP-MS included:

[0074] A single element standard solution of cadmium was precisely measured, and a series of solutions containing Cd at a concentration of 0.2, 0.5, 2, 4, and 10 μg/mL were prepared using a 5% HNO.sub.3 solution, and a standard curve was drawn. The working conditions of ICP-MS are shown in Table 1.

TABLE-US-00004 TABLE 1 Working parameters of ICP-MS instrument Working Item Working Item parameter Plasma gas flow 15.0 L/min Peristaltic pump 0.20 r/s Sampling depth 10 mm Number of 1 repetitions Nebulization 2° C. Auxiliary 0.8 L/min chamber gas flow He gas flow 5 mL/min Carrier 0.8 L/min gas flow Radio- 1550 W Data Peak frequency sampling hopping power mode sampling Determination 6 Number 100 point/peak of scans Number of 3 repetitions

[0075] According to an analysis of fitting degree of the curve, a correlation coefficient of cadmium was determined to be 0.997, with a desirable linear relationship.

[0076] (2) Bioaccessibility of Cadmium in Earthworm Based on In Vitro PBET Method

[0077] 1) Preparation of extracted gastric juice and investigation of a dissolution rate of cadmium in earthworm: earthworm samples were crushed, sieved through a 50-mesh sieve to obtain earthworm powder. To 0.5 g of the earthworm powder was added 50 mL of simulated gastric juice to obtain a mixture. Argon was introduced for 1 min to 2 min to simulate an anaerobic environment in the gastrointestinal tract, the mixture was shaken at 100 r/min for 1 h in a constant-temperature water bath at 37° C., and centrifuged at 4,000 r/min for 5 min; 25 mL of a resulting supernatant was concentrated to 3 mL at low temperature on an electric hot plate, and 5 mL of nitric acid was added to the supernatant after cooling, followed by digestion in a microwave digestion apparatus. After cooling, a resulting digestion solution was made up to a 50 mL in a volumetric flask with ultrapure water for testing; meanwhile, a reagent blank solution was prepared in the same way. The simulated gastric juice was prepared by mixing 1.25 g of pepsin, 0.5 g of sodium citrate, 0.5 g of sodium malate, 420 μL of lactic acid, and 500 μL of acetic acid, making up to 1 L after dissolving, and adjusting pH to a value of 2.0 with hydrochloric acid.

[0078] 2) Preparation of extracted intestinal juice and investigation of a dissolution rate of cadmium in earthworm: after gastric juice extraction, 50 mL of simulated intestinal juice was added to residues to obtain a mixture, the mixture was shaken at 100 r/min for 4 h in a constant-temperature water bath at 37° C., and centrifuged at 4,000 r/min for 5 min; 25 mL of a resulting supernatant was concentrated to 3 mL at low temperature on an electric hot plate, and 5 mL of nitric acid was added to the supernatant after cooling, followed by digestion in a microwave digestion apparatus; after cooling, a resulting digestion solution was diluted to a 50 mL in a volumetric flask with ultrapure water for testing; meanwhile, a reagent blank solution was prepared in the same way. The simulated intestinal juice was prepared by 0.5 g of a pancreatic enzyme and 1.75 g of ammonium cholate, making up to 1 L after dissolving, and adjusting pH to a value of 7.0 with NaOH.

[0079] After the extraction, the residual cadmium was determined in the extracted gastric juice and intestinal juice using the ICP-MS method in step (1) separately to obtain the bioaccessibility of cadmium in earthworm.

[0080] A correlation model was constructed according to results of the relative bioavailability and the bioaccessibility to obtain a regression equation.

EXAMPLE 3

[0081] A method for constructing a correlation model between relative bioavailability and bioaccessibility of cadmium in earthworm included the following steps:

[0082] (1) Determination of Relative Bioavailability of Cadmium in Earthworm

[0083] After adaptive feeding, female mice having a body weight of 18 g to 22 g were randomly grouped (6 mice/group), and weighed after fasting overnight; 10 batches of earthworm groups were given multiple doses of intragastric administration of earthworm; high-concentration, medium-concentration and low-concentration positive control groups were administered with corresponding concentrations of cadmium by gavage, separately; meanwhile, mice administered with normal saline by gavage were used as a blank control group. The grouping and administration were conducted as follows:

[0084] 1) control group: 6 mice were subjected to administration of normal saline at 1 mL/mice by gavage;, once a day;

[0085] 2) low-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 4.5 mg/kg, once a day;

[0086] 3) medium-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 22 mg/kg, once a day;

[0087] 4) high-concentration positive control group: 6 mice were subjected to administration at 1 mL/mice by gavage with a Cd concentration of 105 mg/kg, once a day; and

[0088] 5) earthworm group: 6 mice were subjected to administration at 1 mL/mice by gavage with a earthworm concentration of 7.401 mg/kg, once a day.

[0089] After 6 days of continuous administration, the mice were sacrificed, and liver, kidney, brain, and femur tissues of the mice were taken out, weighed, freeze-dried, and digested; cadmium content of each tissue was determined by ICP-MS, and relative bioavailability was calculated; where the relative bioavailability was calculated according to the following formula:

[00005]$\mathrm{Relative}\mathrm{bioavailability}=\frac{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{Earthworm}}}{\mathrm{Earthworm}\mathrm{concentration}}\times \frac{\mathrm{Positive}\mathrm{control}\mathrm{group}\mathrm{concentration}}{{\left(\mathrm{Liver}+\mathrm{kidney}+\mathrm{brain}+\mathrm{femur}\right)}_{\mathrm{positive}}}$

[0090] where, (liver+kidney+brain+femur).sub.earthworm was the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 6 days after administration of the earthworm group (unit: μg/g dry weight), (liver+kidney+brain+femur).sub.positive was the sum of cadmium content in the liver, kidney, brain and femur tissues of the mice determined 6 days after administration of the low-concentration positive control group, the medium-concentration positive control group, and the high-concentration positive control group (unit: μg/g dry weight).

[0091] The method for determining cadmium by ICP-MS included:

[0092] A single element standard solution of cadmium was precisely measured, and a series of solutions containing Cd at a concentration of 0.2, 0.5, 2, 4, and 10 μg/mL were prepared using a 5% HNO.sub.3 solution separately, and a standard curve was drawn. The working conditions of ICP-MS are shown in Table 1.

TABLE-US-00005 TABLE 1 Working parameters of ICP-MS instrument Working Item Working Item parameter Plasma gas flow 15.0 L/min Peristaltic pump 0.20 r/s Sampling depth 10 mm Number of 1 repetitions Nebulization 2° C. Auxiliary 0.8 L/min chamber gas flow He gas flow 5 mL/min Carrier 0.8 L/min gas flow Radio- 1550 W Data Peak frequency sampling hopping power mode sampling Determination 6 Number 100 point/peak of scans Number of 3 repetitions

[0093] According to analysis of the fitting degree of the curve, a correlation coefficient of cadmium was determined to be 0.997, with a desirable linear relationship.

[0094] (2) Bioaccessibility of Cadmium in Earthworm Based on In Vitro PBET Method

[0095] 1) Preparation of extracted gastric juice and investigation of a dissolution rate of cadmium in earthworm: Earthworm samples were crushed, sieved through a 50-mesh sieve to obtain earthworm powder, 0.5 g of the earthworm powder was added with 50 mL of simulated gastric juice to obtain a mixture. Argon was introduced for 1 min to 2 min to simulate an anaerobic environment in the gastrointestinal tract, the mixture was shaken at 100 r/min for 1 h in a constant-temperature water bath at 37° C., and centrifuged at 4,000 r/min for 5 min; 25 mL of resulting supernatant was concentrated to 3 mL at low temperature on an electric hot plate, and 5 mL of nitric acid was added to the supernatant after cooling, followed by digestion in a microwave digestion apparatus; after cooling, a resulting digestion solution was diluted to a 50 mL in a volumetric flask with ultrapure water for testing; meanwhile, a reagent blank solution was prepared in the same way. The simulated gastric juice was prepared by mixing 1.25 g of pepsin, 0.5 g of sodium citrate, 0.5 g of sodium malate, 420 μL of lactic acid, and 500 μL of acetic acid, making up to 1 L after dissolving, and adjusting pH to a value of 2.0 with hydrochloric acid.

[0096] 2) Preparation of extracted intestinal juice and investigation of a dissolution rate of cadmium in earthworm: after gastric juice extraction, 50 mL of a simulated intestinal juice was added to residues to obtain a mixture, the mixture was shaken at 100 r/min for 4 h in a constant-temperature water bath at 37° C., and centrifuged at 4,000 r/min for 5 min; 25 mL of a resulting supernatant was concentrated to 3 mL at low temperature on an electric hot plate, and 5 mL of nitric acid was added to the supernatant after cooling, followed by digestion in a microwave digestion apparatus; after cooling, a resulting digestion solution was diluted to a 50 mL in a volumetric flask with ultrapure water for testing; meanwhile, a reagent blank solution was prepared in the same way. The simulated intestinal juice was prepared by mixing 0.5 g of a pancreatic enzyme and 1.75 g of ammonium cholate, making up to 1 L after dissolving, and adjusting pH to a value of 7.0 with NaOH.

[0097] After the extraction, the residual cadmium was determined in the extracted gastric juice and intestinal juice using the ICP-MS method in step (1) separately to obtain the bioaccessibility of cadmium in earthworm.

[0098] A correlation model was constructed according to results of the relative bioavailability and the bioaccessibility to obtain a regression equation.

[0099] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.